Subset of Hybrid Eukaryotic Proteins Is Exported by the Type I Secretion System of Erwinia chrysanthemi

Abstract

Erwinia chrysanthemi exports degradative enzymes by using a type I protein secretion system. The proteases secreted by this system lack an N-terminal signal peptide but contain a C-terminal secretion signal. To explore the substrate specificity of this system, we have expressed the E. chrysanthemi transporter system (prtDEF genes) in Escherichia coli and tested the ability of this ABC transporter to export hybrid proteins carrying C-terminal fragments of E. chrysanthemi protease B. The C terminus contains six glycine-rich repeated motifs, followed by two repeats of the sequences DFLV and DIIV. Two types of hybrid proteins were assayed for transport, proteins with the 93-residue-protease-B C terminus containing one glycine-rich repeat and both hydrophobic terminal repeats and proteins with the 181-residue C terminus containing all repeat motifs. Although the shorter C terminus is unable to export the hybrids, the longer C terminus can promote the secretion of hybrid proteins with N termini as large as 424 amino acids, showing that the glycine-rich motifs are required for the efficient secretion of these hybrids. However, the secretion of hybrids occurs only if these proteins do not carry disulfide bonds in their mature structures. These latter results suggest that disulfide bond formation can occur prior to or during the secretion. Disulfide bonds may prevent type I secretion of hybrids. One simple hypothesis to explain these results is that the type I channel is too narrow to permit the export of proteins with secondary structures stabilized by disulfide bonds.Nonpathogenic strains of Escherichia coli, such as the laboratory strain E. coli K-12, export few proteins to the external medium (53). The secretion of proteins in E. coli depends on a type II (Sec-dependent) mechanism in which unfolded proteins carrying an N-terminal signal sequence are transported across the inner membrane to the periplasm and then processed and folded in the periplasm prior to their translocation across the outer membrane. To initiate type II secretion, it is thought that a polypeptide and its signal peptide must be fully extended to cross the inner membrane (3). After crossing the inner membrane, proteins are folded in the periplasm in a process assisted by chaperones. In addition, the periplasm contains the enzymes required for the correct assembly of disulfide bonds to complete protein folding (5, 48).In contrast, many gram-negative pathogens including enteropathogenic E. coli (38), Yersinia spp. (36), Salmonella enterica serovar Typhimurium (39), and Vibrio cholerae (35) export virulence factors required for host colonization and survival. These virulence factors are secreted by mechanisms that operate independently from the type II system, mechanisms that involve specialized membrane transport apparatuses (64). A subset of virulence factors is exported by a type I mechanism in which three proteins assemble to form a transmembrane structure that couples the export of protein substrates with ATP hydrolysis. These ABC transporters in...

Calcium-induced Folding and Stabilization of the Pseudomonas aeruginosa Alkaline Protease

“…First, based on the apparent affinities and activities of the RTX and AP proteins, AP would remain unfolded or destabilized within the cell. In an extended conformation, transport could occur without the need for a cellular unfoldase activity because secretion is thought to occur post-translationally and require an unfolded polypeptide substrate (21,27). Maintaining the cytosolic AP in an unfolded conformation would also limit unwanted enzymatic activity prior to secretion.…”

Family I.3 lipase: bacterial lipases secreted by the type I secretion system

“…'Natural' passenger proteins of the T1SS do not usually contain any cysteine residue, or in some cases, contain only one cysteine residue, indicating that these proteins do not have any intramolecular disulfide bond. A study on secretion of hybrid eukaryotic proteins fused with C domain of E. chrysanthemi protease showed that E. chrysanthemi T1SS can only secrete proteins that do not have any intramolecular disulfide bond [79]. Another report, however, showed that E. coli alkaline phosphatase, a dimeric periplasmic protein with two intramolecular disulfide bonds (four cysteine residues in one monomer), can be secreted extracellularly when it is fused with the C-terminal secretion signal of α-hemolysin [80].…”

“…AF083061) [59]. In this review, we use the latter as the correct sequence, since the first sequence contains nine cysteine residues, a feature uncommon in proteins secreted via the T1SS [79].…”

Multiple Signals Direct the Assembly and Function of a Type 1 Secretion System

“…However, previous studies pointed out that additional domains must be involved in the interaction with the secretion complex. First, efficient secretion of heterologous proteins fused to E. coli HlyA or E. chrysanthemi PrtB requires C-terminal fragments much larger than the minimal secretion signals (20,25,33). Second, hemophore HasA(1-174) without its C-terminal signal retains its capacity to interact with HasD and induce the assembly of the tripartite complex (7).…”